Abstract
The human coagulation protease factor VII plays a pivotal role in the initiation of the coagulation cascade by both the extrinsic and the intrinsic pathway. Although the gene, encoding factor VII, is expressed predominantly in the liver, the mechanisms underlying this tissue-specific expression have not been elucidated. In this study, we have analyzed the contribution of 5 kilobases upstream of the ATG translational initiation codon upon hepatic factor VII gene transcription. Transient transfection assays of a set of nested deletions in both liver and non-liver cell lines, HepG2 and HeLa respectively, indicate that several regions are involved in liver-specific expression. A slight negative effect on factor VII promoter activity in HepG2 cells is mediated by sequences upstream of position -1212. DNase I protection experiments reveal six footprints, FPVII1 through FPVII6, within the proximal 714 base pairs but a minimal promoter of 165 base pairs containing only FPVII3-6 is sufficient to confer liver-specific expression in HepG2 cells. Interestingly, FPVII6, at position -14 to +10 on the sense strand, would indicate that an as yet unknown transcription factor covers the ATG translational initiation codon. Gel retardation experiments show that the liver-enriched transcription factor HNF-4 binds specifically to footprint FPVII4 at position -71 to -49. Furthermore, a T --> A transversion, that in the HNF-4 binding site of factor IX causes a severe bleeding disorder, was introduced into the HNF-4-binding site of factor VII and reduced promoter activity by 20-50%. Coordinate HNF-4-mediated regulation of several blood protease genes as well as genes involved in lipid metabolism might account for the positive correlation of these factors with increased risk of occlusive heart diseases.
Highlights
A positive correlation between high plasma factor VII antigen levels (FVIIag), procoagulant activity (FVIIc), and an increased risk of coronary heart disease has been demonstrated [5]
Sequence alignment of the factor VII and factor X promoters revealed a similarity of 86% for a small 37-bp element located in factor VII promoter at position Ϫ2340 to Ϫ2304 and in the factor X promoter in the same orientation at position Ϫ520 to Ϫ485
The regulation of factor IX gene expression is of particular interest, since the Leyden phenotype of the severe bleeding disorder hemophilia B is caused by several point mutations within the promoter region, of which a number map in the HNF-4-binding site, reducing the affinity of HNF-4 for the promoter
Summary
A positive correlation between high plasma factor VII antigen levels (FVIIag), procoagulant activity (FVIIc), and an increased risk of coronary heart disease has been demonstrated [5]. Males heterozygous for an Arg353 to Gln353 polymorphism found in about 20% of the caucasian population have FVIIag concentrations reduced by 20 –25%, which presumably reduces the risk of coronary heart disease and thrombosis by lowering the proportion of factor VII molecules being in the activated state [11, 12]. From these prospective studies it is possible that a drug that interferes with factor VII expression levels could be an antithrombotic, and the first step toward such a drug would be to understand the regulatory mechanisms of factor VII expression. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U14580
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